PROJECT SUMMARY Orthosomycins are a family of potent antibiotic oligosaccharides that target a wide spectrum of gram-positive bacteria, including most antibiotic-resistant strains. It is less appreciated that subsets of orthosomycins also target gram-negative drug-resistant bacteria, including members of the Enterobacteriaceae family, which are ranked as some of the highest threats to human health by the Centers for Disease Control and Prevention. Orthosomycins demonstrate high potency, good bioavailability, and low toxicity in vivo in both animals and humans. The promise of this class was explored preclinically, and through subsequent development of one orthosomycin, everninomicin A (Ziracin). Despite Ziracin?s advancement to phase III clinical trials, unstated pharmacological complications led to a strategic decision to discontinue clinical development of this scaffold in 2000. In the intervening time, no attempts have been made to improve the orthosomycins. We speculate that addressing pharmacological liabilities was complicated due to the unknown reasons for withdrawal, the fragmentary understanding of the everninomicin molecular target, and the challenges inherent in orthosomycin chemical synthesis, which requires at least 130 steps. Recently, the structures of orthosomycins bound in the bacterial ribosome have been solved, revolutionizing our understanding of their molecular target and mechanism of action and creating opportunities to improve ribosome interactions and pharmacological properties via targeted structural changes. Contemporaneously, we developed a set of genetic tools for editing the genome of the producing organisms, as well as advanced the understanding of the biochemical mechanisms and pathways of orthosomycin assembly. We have initiated, but not completed, an exploration of the formation of the interglycosidic orthoester linkages, the formation and attachment of dichloroisoeverninic acid, and the biosynthesis of the eurekanate sugar, unique to orthosomycin antibiotics. This convergence of progress in the understanding of orthosomycin biosynthesis and target identification provides an unprecedented opportunity to address the complications limiting the clinical utility of these molecules by improving their potency and pharmacological properties. To further this goal, our specific aims are to (1) characterize and tune the interactions of orthosomycins with rProtein uL16, (2) investigate h89 and h91 spanning interactions of orthosomycins, and (3) develop access to unnatural orthosomycin analogs with targeted structural changes impacting the rRNA h91 pocket. Premise: In this project, we outline probable origins and solutions to pharmacological liabilities. Leveraging biosynthetic insights, we will generate targeted changes in the scaffolds of orthosomycins using a combined genetic, chemical, and biochemical approach. With these designed variants, we will determine the extent to which structural variations can improve ribosome binding, as well as modify pharmacological properties to remove liabilities and improve the therapeutic index.